Abstract Understanding retinal metabolism in detail is fundamental to knowing how the retina withstands, compensates for, or suffers from the stresses imposed by systemic changes and disease. This application takes a new approach to understanding glycolytic and oxidative metabolism of the mammalian retina, by recording spatial profiles of oxygen and pH in the isolated rat and mouse retina with microelectrodes, using mathematical models of diffusion to extract information about rates of substrate utilization and waste generation, and employing pharmacology to isolate different processes. This strategy is complimentary to previous in vivo and in vitro approaches, but allows a separation of metabolic events in the inner and outer retina, which has rarely been possible, and cannot be done with measurements of whole tissue metabolism. Microelectrode approaches simultaneously allow recording of transretinal and intraretinal electroretinograms to monitor retinal function. There are three aims. 1) Depth profiles of oxygen will be recorded in order to determine quantitatively how the metabolism of the isolated retina compares to that in vivo, and how the inner and outer retina differ metabolically. Photoreceptors are known to perform high rates of (anaerobic) glycolysis, but the balance between oxidative and glycolytic energy production is not known in the inner retina, and whether this changes depending on glucose supply. Retinal blood flow increases in response to flickering light (neurovascular coupling), but the size of the metabolic change in the inner retina that drives this is unknown, and will be measured here. 2) By recording depth profiles of pH in the isolated retina, the production of acidic waste will be quantified. Glycolysis and oxidative metabolism are very different in acid production. To identify the components of acid production, hypoxia and metabolic poisons that suppress either glycolysis or oxidative metabolism will be used. 3) In some tissues there is evidence that glycolysis and oxidative metabolism are compartmentalized, with a transfer of lactate and/or pyruvate from glycolytically active cells to ones that depend more on oxidative metabolism. This concept has led to the idea that the retina uses a similar strategy, with lactate being produced in Muller cells and shuttled to neurons. The relatively recent availability of selective blockers of monocarboxylate transport, provides an opportunity to evaluate the importance of such transfer quantitatively in both the inner and outer retina. All the information to be gained is fundamental to understanding how the retina changes in disease, and what the capabilities of the inner and outer retina are for oxidative and glycolytic metabolism under different conditions.